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While the branched DNA (bDNA) assay is an established bioanalytical method for measurement of lipid nanoparticle (LNP)-encapsulated messenger RNA (mRNA) pharmacokinetic parameters, reverse transcription-quantitative polymerase chain reaction (RT-qPCR) has been considered as an alternative platform. RT-qPCR and bDNA platforms were compared for sensitivity, specificity, correlation, and overall assay performance using serum and tissue samples from 2 nonclinical mouse studies of a therapeutic mRNA candidate, LNP-PAH-mRNA, which encodes for human phenylalanine hydroxylase enzyme. Pharmacokinetic parameter noncompartmental analysis was completed using Phoenix WinNonlin. The assays were compared using simple linear regression and Bland-Altman analyses. Sensitivity ranged from 0.05 to 6.40 ng/mL for the bDNA assays, from 0.00000761 to 7.61 ng/mL in serum, and from 0.000179 to 179 ng/g in tissue for the RT-qPCR assay. Inter-assay accuracy was within ± 10%, inter-assay precision was ≤ 10%, and the total error for both assays was ≤ 20%. RT-qPCR serum mRNA concentrations were 2- to fourfold lower compared with the bDNA assay, whereas tissue samples were comparable between assays. A linear relationship with - 0.37 to - 0.02 systematic bias demonstrated acceptable concordance. Bland-Altman plots demonstrated close equivalence, with a negative bias of < 0.5, and ≥ 95% of the data points were within the 95% limits of agreement. The comparison of the RT-qPCR with bDNA assay platforms for quantification of pharmacokinetic properties of an mRNA-LNP therapeutic has demonstrated acceptable concordance. This comparison reinforces the use of the RT-qPCR, a widely accessible strategy, as an alternative platform for the quantification of subsequent mRNA-LNP therapeutics., Competing Interests: Declarations. Conflict of Interest: J.O., J.P., L.C., L.B., R.B., D.J., and J.M. are employees of Moderna, Inc. and hold stock/options in the company., (© 2024. The Author(s), under exclusive licence to American Association of Pharmaceutical Scientists.)

Immunogenicity testing and characterization is an important part of understanding the immune response to administration of a protein therapeutic. Neutralizing antibody (NAb) assays are used to characterize a positive anti-drug antibody (ADA) response. Harmonization of reporting of NAb assay performance and results enables efficient communication and expedient review by industry and health authorities. Herein, a cross-industry group of NAb assay experts have harmonized NAb assay reporting recommendations and provided a bioanalytical report (BAR) submission editable template developed to facilitate agency filings. This document addresses key bioanalytical reporting gaps and provides a report structure for documenting clinical NAb assay performance and results. This publication focuses on the content and presentation of the NAb sample analysis report including essential elements such as the method, critical reagents and equipment, data analysis, study samples, and results. The interpretation of immunogenicity data, including the evaluation of the impact of NAb on safety, exposure, and efficacy, is out of scope of this publication., (© 2024. The Author(s).)

Evolving immunogenicity assay performance expectations and a lack of harmonized neutralizing antibody validation testing and reporting tools have resulted in significant time spent by health authorities and sponsors on resolving filing queries. A team of experts within the American Association of Pharmaceutical Scientists' Therapeutic Product Immunogenicity Community across industry and the Food and Drug Administration addressed challenges unique to cell-based and non-cell-based neutralizing antibody assays. Harmonization of validation expectations and data reporting will facilitate filings to health authorities and are described in this manuscript. This team provides validation testing and reporting strategies and tools for the following assessments: (1) format selection; (2) cut point; (3) assay acceptance criteria; (4) control precision; (5) sensitivity including positive control selection and performance tracking; (6) negative control selection; (7) selectivity/specificity including matrix interference, hemolysis, lipemia, bilirubin, concomitant medications, and structurally similar analytes; (8) drug tolerance; (9) target tolerance; (10) sample stability; and (11) assay robustness., (© 2023. The Author(s).)

A cell-based assay was developed to detect neutralizing anti-drug antibodies (NAbs) against odronextamab, a CD20xCD3 bispecific monoclonal antibody (mAb) under investigation for treatment of CD20+ B cell malignancies. In this assay, odronextamab bridges between two cell types, CD20-expressing HEK293 cells and CD3-expressing Jurkat T cells that generate a luciferase signal upon CD3 clustering. Patient samples containing NAbs directed to either arm of the bispecific drug block the odronextamab bridge formation between the cell lines thus preventing the generation of the luciferase signal. We determined that other anti-CD20 therapeutics also block bridge formation, resulting in false-positive results. In patient samples from odronextamab clinical trials, approximately 30% of baseline samples had a strong false-positive NAb signal that correlated with the presence of prior rituximab (anti-CD20) therapy. We determined that rituximab interference can be minimized by the addition of anti-rituximab antibodies in the NAb assay. Understanding and mitigating the impact of prior biologic exposure is increasingly important for implementing a successful bioanalytical strategy to support clinical drug development, especially in the immuno-oncology field. Odronextamab neutralizing antibody assay, interference, and mitigation. A Design of the odronextamab neutralizing antibody (NAb) assay where anti-CD20xCD3 drug bridges between CD20-expressing HEK293 cells and Jurkat T cells expressing an NFAT response element and luciferase reporter. True NAb prevents odronextamab from bridging between target and effector cells, thus preventing the expression of luciferase. B Interference with odronextamab from other anti-CD20 therapeutic antibodies (e.g., rituximab) from prior disease treatment generates a false-positive NAb result. Assay interference can be mitigated with an anti-idiotypic antibody against the interfering therapy., (© 2022. The Author(s).)

A robust, quantitative method for assessing the neutralizing capacity of anti-therapeutic protein antibodies was developed and tested using 4 analytical assay platforms typically used for detection of anti-drug neutralizing antibodies. The method described here utilized titration of increasing concentrations of therapeutic protein into serum containing anti-therapeutic protein antibodies, either positive control antibodies or clinical samples. Neutralizing capacities were calculated by determining the EC50 from the titration curves. The neutralizing capacity of purified anti therapeutic protein antibodies was expressed in terms of "μg of drug neutralized per μg of anti-TP antibody" present. In the case of serum originating from clinical study subjects, the neutralizing capacity of the samples was expressed as "μg of drug neutralized per mL of serum". A relative shift in EC50 values was observed as the amount of serum or antibody was changed resulting in a proportional shift of the calculated neutralizing capacity. Application of this approach using different assay platforms was consistent providing evidence for its potential to be a useful approach to characterize, qualify and compare neutralizing positive control antibody preparations or clinical samples. Using this methodology, we were able to draw a clear correlation between the neutralizing capacity and the effect of these antibodies on a clinical pharmacodynamic (PD) marker. Determination of the neutralizing capacity of antibody positive samples from subjects in a clinical study indicated direct correlation of pharmacodynamic results with non-response, partial response and response to high, mid and low neutralizing capacities respectively., (Copyright © 2014 Elsevier B.V. All rights reserved.)

The method described here employs a high-content cell-based assay format for the detection of neutralizing antibodies (NAbs) to panitumumab, a fully human monoclonal antagonistic antibody to the human epidermal growth factor (EGF) receptor in human serum (screening assay). A specificity assay was also developed and qualified to confirm that the neutralizing activity was attributable to the presence of NAbs and not due to serum interference (serum interference assay). The ArrayScan IV HCS reader was used for the measurement of tyrosine phosphorylation of the epidermal growth factor receptor (EGFR) and STAT-1 redistribution between the cytoplasm and nucleus in the human epidermoid carcinoma cell line A431. Assay conditions were developed by (1) optimizing the response of the A431 cells to recombinant human EGF in pooled human serum, (2) evaluating the ability of panitumumab to inhibit the EGF response, and (3) assessing the assay's sensitivity for detecting a positive control affinity purified rabbit polyclonal anti-panitumumab antibody. Panitumumab dose-dependently inhibited 4 ng/mL EGF, and the positive control antibody showed a dose-dependent neutralization of 50 ng/mL panitumumab. The experiments indicated that in comparison to STAT-1 translocation, EGFR phosphorylation was the optimal endpoint for the screening and serum interference assays. Assay cut points were derived for the screening and serum interference assays by obtaining normalized ratios of mean fluorescence intensity values obtained with EGFR phosphorylation by testing sera from healthy human donor sera. The assay sensitivity was determined to be 0.125 microg/mL for the positive control antibody.